Fibroblast-like synoviocytes from rheumatoid arthritis patients express functional IL-15 receptor complex: endogenous IL-15 in autocrine fashion enhances cell proliferation and expression of Bcl-x(L) and Bcl-2

The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of fibroblast-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and growth factors. Because IL-15 exerts properties of antiapoptotic and growth factors, and is produced by RA FLS, we hypothesized that IL-15 participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-15R. Indeed, RA FLS express IL-15Ralpha at mRNA and protein levels. Moreover, we confirmed the presence of IL-2Rbeta and common gamma-chains. Interestingly, TNF-alpha or IL-1beta triggered significant elevation of IL-15Ralpha chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-15 on Bcl-2 and Bcl-x(L) expression, FLS proliferation, and apoptosis. Exogenous IL-15 enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-x(L). To test the role of endogenous IL-15 in the activation of RA FLS, an IL-15 mutant/Fcgamma2a protein exerting properties of specific antagonist to the IL-15Ralpha chain was used. We found that blocking IL-15 biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-x(L), and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-15Rs by FLS-derived IL-15.     

Absence of the CD1 molecule up-regulates antitumor activity induced by CpG oligodeoxynucleotides in mice

The role of NKT cells on antitumor activity of CpG oligodeoxynucleotides (ODNs) was evaluated by peritumoral injections of CpG-ODNs in s.c. melanoma-bearing mice of strains differing in the number of NKT cells (athymic nude mice, recombination-activating gene(-/-)/transgenic V(alpha)14/Vbeta8.2 mice that generate NKT cells; J(alpha)281(-/-) mice and CD1(-/-) mice, which both have a strongly reduced number of NKT cells; and C57BL/6 wild-type mice). Tumor growth was significantly inhibited in strains enriched or depleted of NKT cells. The two murine strains having a reduced number of NKT cells differed significantly in the CpG-dependent tumor growth inhibition: in J(alpha)281(-/-) mice this inhibition was superimposable to that observed in C57BL/6 mice, while in CD1(-/-) mice the inhibition was dramatic. The increased tumor inhibition in CD1(-/-) correlated with a significantly higher ratio of IFN-gamma-IL-4 production in response to CpG as compared with C57BL/6 and J(alpha)281(-/-) mice. Experiments in which preparations of APCs and lymphocytes of the three strains were mixed showed that in the presence of APCs not expressing CD1, the production of CpG-ODN-induced type 1 cytokines was higher. Phenotype analysis of IFN-gamma- and IL-4-producing cells revealed that the differences between CD1(-/-) and C57BL/6 in the production of these two cytokines were mainly due to CD3(+) T lymphocytes. These data point to a regulatory role for the CD1 molecule in antitumor activity induced by danger signals, independently of V(alpha)14 NKT cells. The identification of a CD1-dependent suppressive subpopulation(s) might have important implications for the study of tolerance in the context of cancer, autoimmunity, and transplantation.     

A cellular tensegrity model to analyse the structural viscoelasticity of the cytoskeleton

This study describes the viscoelastic properties of a refined cellular-tensegrity model composed of six rigid bars connected to a continuous network of 24 viscoelastic pre-stretched cables (Voigt bodies) in order to analyse the role of the cytoskeleton spatial rearrangement on the viscoelastic response of living adherent cells. This structural contribution was determined from the relationships between the global viscoelastic properties of the tensegrity model, i.e., normalized viscosity modulus (eta(*)), normalized elasticity modulus (E(*)), and the physical properties of the constitutive elements, i.e., their normalized length (L(*)) and normalized initial internal tension (T(*)). We used a numerical method to simulate the deformation of the structure in response to different types of loading, while varying by several orders of magnitude L(*) and T(*). The numerical results obtained reveal that eta(*) remains almost independent of changes in T(*) (eta(*) proportional, variant T(*+0.1)), whereas E(*) increases with approximately the square root of the internal tension T(*) (from E(*) proportional, variant T(*+0.3) to E(*) proportional, variant T(*+0.7)). Moreover, structural viscosity eta(*) and elasticity E(*) are both inversely proportional to the square of the size of the structure (eta(*) proportional, variant L(*-2) and E(*) proportional, variant L(*-2)). These structural properties appear consistent with cytoskeleton (CSK) mechanical properties measured experimentally by various methods which are specific to the CSK micromanipulation in living adherent cells. Present results suggest, for the first time, that the effect of structural rearrangement of CSK elements on global CSK behavior is characterized by a faster cellular mechanical response relatively to the CSK element response, which thus contributes to the solidification process observed in adherent cells. In extending to the viscoelastic properties the analysis of the mechanical response of the cellular 30-element tensegrity model, the present study contributes to the understanding of recent results on the cellular-dynamic response and allows to reunify the scattered data reported for the viscoelastic properties of living adherent cells.     

Hepatitis C virus-like particle morphogenesis

Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.     

Analysis of xanthophore and pterinosome biogenesis in zebrafish using methylene blue and pteridine autofluorescence

We have identified two simple methods to analyse xanthophore and pterinosome biogenesis in zebrafish. The first uses methylene blue (methylthionium chloride), a redox dye which specifically labels xanthophores and pterinosomes, while the second uses autofluorescence to detect pteridine levels; these methods may be used to detect the number, location and shape of xanthophores and pterinosomes. These assays were applied to two zebrafish mutants--brie and yobo--and revealed that both mutants have pterinosome biogenesis and pteridine synthesis defects. Additionally, using capillary electrophoresis, we provide evidence that sepiapterin is responsible for the yellow colour and blue-light induced fluorescence in zebrafish embryos.     

Synthesis,radiolabeling and preliminary biological evaluation of radiolabeled 5-methyl-6-nitroquipazine,a potential radioligand for the serotonin transporter

5-Methyl-6-nitroquipazine, a novel analogue of the potent and selective serotonin transporter inhibitor 6-nitroquipazine was synthesized and radiolabeled with tritium and the positron emitter carbon-11. [³H]5-methyl-6-nitroquipazine was found to have a K_d=51±7 pM. The high affinity and the facile labeling of [¹¹C]5-methyl-6-nitroquipazine makes it a promising radioligand for visualization of the serotonin transporter with positron emission tomography.     

Bilayered clathrin coats on endosomal vacuoles are involved in protein sorting toward lysosomes

In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron microscopy we show that the coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes AP1, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. No association of endosomal coated areas with tracks of tubulin or actin was found. By quantitative immunoelectron microscopy, we found that the lysosomal-targeted receptors for growth hormone (GHR) and epidermal growth factor are concentrated in the coated membrane areas, whereas the recycling transferrin receptor is not. In addition, we found that the proteasomal inhibitor MG 132 induces a redistribution of a truncated GHR (GHR-369) toward recycling vesicles, which coincided with a redistribution of endosomal vacuole-associated GHR-369 to the noncoated areas of the limiting membrane. Together, these data suggest a role for the bilayered clathrin coat on vacuolar endosomes in targeting of proteins to lysosomes.     

Ecology,development and pathogenicity of Buddenbrockia plumatellae Schröder, 1910 (Myxozoa,Malacosporea) (syn. Tetracapsula bryozoides) and establishment of Tetracapsuloides n. gen. for Tetracapsula bryosalmonae

Buddenbrockia plumatellae, an enigmatic worm-like myxozoan, was observed as continuously writhing free and attached 'worms' and as free mature spores in the coelom of the freshwater bryozoans Plumatella fungosa, Hyalinella punctata, and Fredericella sp. 'Worm' numbers could double every three days. 'Worms' and spores could be expelled from colonies by external pressure. Some mature 'worms' exited actively, entraining release of free spores, and gradually ceased movement outside the host. Bryozoans sealed off infected regions of the colony. Infected colonies grew slowly, produced no statoblasts, and eventually regressed and died. Transmission was not achieved and prevalence was low. Electron microscopy of 'worms' revealed a single layer of mural cells on a fibrous basal lamina overlying four longitudinal muscle blocks and an inner sheet of two types of proliferating cells, an organization indicative of the bilaterian ancestry of the Myxozoa. Primary type A cells were attached directly by striated tubules to mural cells at positions between muscle blocks. Secondary type A cells had a secretory function. Type B cells underwent meiosis and subsequently developed to typical malacosporean myxozoan spores filling the internal cavity of the 'worms'. External tubes were formed during capsulogenesis in 'worms' from Fredericella sp. Tetracapsula bryozoides is synonymised with Buddenbrockia plumatellae and a new genus is proposed for Tetracapsula bryosalmonae.     

Wild-type intracellular bacteria deliver DNA into mammalian cells

Gene transfer in vitro from intracellular bacteria to mammalian phagocytic and non-phagocytic cells and in vivo in mice has been reported. The bacteria used as DNA delivery vectors were engineered to lyze upon entry in the cell due to impaired cell wall synthesis for Shigella flexneri and invasive Escherichia coli , or production of a phage lysin for Listeria mono- cytogenes . In vivo gene transfer was obtained with attenuated Salmonella typhimurium and resulted in stimulation of mucosal immunity. We report that wild-type intracellular human pathogens, such as L. monocytogenes EGD or LO28 and S. flexneri M90T, mediate efficient in vitro transfer of functional genes into epithelial and macrophage cell lines. A low- efficiency transfer was obtained from strain EGD to mouse peritoneal macrophages. DNA transfer with S. typhimurium was observed only from atten-uated aroA strain SL7207 into COS-1 cell line. As demonstrated by the study of listeriolysin-defective L. monocytogenes or of S. typhimurium SL7207 aroA engineered to secrete listeriolysin, escape of bacteria or of plasmid DNA from the intracytoplasmic vacuole is required for transfer of genetic information to occur.